Shock, which is induced by endotoxin, is known as septic shock (SS). This condition is a life-threatening situation which occurs following infections by Gram-negative bacteria as complication of surgery, prolonged hospitalization, accidents and other traumatic events. It is today well recognized that the agent responsible for this disease is the bacterial endotoxin, a glycolipid antigen present only on the surface of Gram-negative bacteria. This glycolipid is also known as lipo-poly saccharide (LPS) or lipo-oligosaccharide (LOS) depending from the size of the carbohydrate chain which is covalently bound to the fatty-acid-rich moiety called Lipid A (LipA). Only Lipid A is responsible of the major toxic effects shown by endotoxin (LPS). Once endotoxin is released in the blood-stream by bacteria, specialized cells of the immune system like macrophages and monocytes are activated by the endotoxin and several immune mediators are released (Cytokines such as Interleukin-1 and Interleukin-6; .alpha.--Tumor necrosis factor; .gamma.--Interferon). Furthermore, endotoxin also activates the complement cascade which results in cell lysis with the consequent release of proteolytic enzymes promoting the release of vasoactive effectors from platelets (e.g.: bradykinine and histamine). The final result is death of the patient in 40-60% of the cases within 48-72 hours. So far, there has been no specific cure or therapy available although bolus injections of adrenal corticosteroids such as methylprednisolone are used.
Polymyxin "B" is known as a molecule that binds and detoxifies bacterial endotoxins and can prevent septic shock when given therapeutically in animal models. However, Polymyxin "B" is a toxic product in vitro and in vivo and this fact limits its potential as a therapeutic agent for the treatment of septic shock.
Septic shock can be caused by infection with any bacteria that cause the release of LPS. These bacteria include Pseudomonas aeroqinosa, Escherichia coli, Salmonella typhi, Neisseria meningitidis, Neisseria gonorrheae, Bordetella pertussis, Klebsiella pneumoniae and the like.
The reasons leading to the reported toxicity of Polymyxin B are not completely understood but they are most likely related to the peculiarity of its amino acid composition, specifically for the content of L.alpha.--.gamma.--, diamino butyric acid (DAB) (49.1% w/w of the structure) which is an analog of the aa Lysine (reported in literature as able to substitute Lysine in the protein synthesis) and for the presence of D-Phenylalanine an isomer of the naturally occurring L-Phenylalanine. Other possible reasons, still related to the aa composition, could be related to the high stability of Polymyxin "B" to proteolytic enzymes as well as to the possible binding to cell receptors structurally comparable to the Lipid A moiety of LPS (the gangliosides of the nervous tissues are glycolipids with N,O--acyl (C.sub.14 -C.sub.18) chains closely related to the N,O--acyl chains present in the Lipid A structure).
The applicants have discovered new conformational peptides that are structurally different from Polymyxin (in virtue of their amino acid composition) but are capable of binding to the same binding site within Lipid A of endotoxins (LOS and LPS) that Polymyxin "B" will also bind. The relative binding efficiency of the new peptides is comparable to the affinity constant value of Polymyxin "B". The complex formed when Lipid A or LPS are reacted with the peptides of the invention is non-toxic and the natural antigenicity of Lipid A and LPS is maintained.
As a consequence of this high-affinity binding to the Lipid A moiety of endotoxins, most of the synthetic peptide analogs have shown the ability to detoxify endotoxins as evidenced by in vitro as well as in vivo analysis. The in vitro test used, as measure of detoxification, the inhibition of the enzymatic cascade leading to the coagulation of the Lymulus lysate (LAL test) by endotoxin. The LAL test is recognized as the most sensitive and predictive test for the toxic and pyrogenic activity of LPS, since pyrogenicity in vivo is related to the release of the endogenous immune modulators Interleukin-1 (IL-1) and alfa-Tumor necrosis factor (.alpha.-TNF), the mediators responsible for the fatalities associated to septic shock. As an in vivo test confirming detoxification of LPS, was then used the Rabbit pyrogen test performed according to the United States Pharmacopeia XXI.
This discovery thus provides a new class of compounds that may be used in the treatment of septic shock. It is anticipated that the new peptides will not exhibit in humans the toxic effects of Polymyxin "B", in virtue of their completely natural amino acid composition as well as for their limited resistance to proteolytic degradation in human serum.
Accordingly, it is a primary object of the invention to provide novel prophylactic and therapeutic agents which may be used in the treatment of septic shock.
It is also an object of this invention to provide novel peptide compounds which may be used in the treatment of septic shock.
It is also an object of this invention to provide novel pharmaceutical compositions which may be used in the treatment of septic shock.
It is also an object of this invention to provide novel complexes of Lipid-A or LPS and a peptide which are antigenic and non-toxic.
It is also an object of this invention to provide a method of producing novel non-toxic Lipid A or LPS antigens.
Conditions other than septic shock where an endotoxin is produced may also be treated by the peptides of the invention using the same dose of peptides which is used to treat septic shock. These conditions include pertussis bacterial meningitis and viral HIV-related infections.
These and other objects of the invention will become apparent from a review of the present specification.